veterinary microbiology diagnostics, swine dysentery draft · america and the emergence of...

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A review of the current state of antimicrobial susceptibility

test methods for Brachyspira

Journal: Canadian Journal of Microbiology

Manuscript ID cjm-2016-0756.R1

Manuscript Type: Review

Date Submitted by the Author: 09-Feb-2017

Complete List of Authors: Kulathunga, Dharmasiri; Western College of Veterinary Medicine, Veterinary Microbiology Rubin, Joseph E.; University of Saskatchewan, Veterinary Microbiology

Keyword: Brachyspira, Susceptibility test, Method standardization, Veterinary diagnostics, Swine dysentery

https://mc06.manuscriptcentral.com/cjm-pubs

Canadian Journal of Microbiology

Draft

A review of the current state of antimicrobial susceptibility test methods for Brachyspira

D.G.R. S. Kulathunga1, J.E. Rubin

1*

1Department of Veterinary Microbiology, University of Saskatchewan, Saskatoon, Canada

D.G.R.S. Kulathunga - [email protected]

J.E. Rubin - [email protected]

*Corresponding Author

52 Campus Drive

Department of Veterinary Microbiology

Phone: (306) 966-7246

Fax: (306) 966-7244

Email: [email protected]

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Abstract

The re-emergence of swine dysentery (Brachyspira-associated muco-haemorrhagic colitis) since

the late 2000's has illuminated diagnostic challenges associated with this genus. The methods

used to detect, identify and characterize Brachyspira from clinical samples have not been

standardized and laboratories frequently rely heavily on in-house techniques. Particularly

concerning is the lack of standardized methods for determining and interpreting the antimicrobial

susceptibility of Brachyspira spp. The integration of laboratory data into a treatment plan is a

critical component of prudent antimicrobial usage, the lack of standardized methods is therefore

an important limitation to the evidence based use of antimicrobials. This review will focus on

describing the methodological limitations and inconsistencies between current susceptibility

testing schemes employed for Brachyspira, provide an overview of what we do know about the

susceptibility of these organisms and suggest future directions to improve and standardize

diagnostic strategies.

Keywords: Brachyspira, susceptibility test, method standardization, veterinary diagnostics,

swine dysentery

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1.0 Introduction

Brachyspira are a genus of Gram-negative, oxygen-tolerant anaerobic spirochetes which

colonize the intestinal tracts of wild and domestic animals and humans (Hampson and Ahmed

2009). Seven species with standing in nomenclature are currently recognized within the genus

Brachyspira, including the causative agents of swine dysentery and porcine, avian and human

intestinal spirochetosis. Swine dysentery, characterized by profuse muco-hemorrhagic colitis,

was first described in 1921 in the United States, although the causative agent (B. hyodysenteriae)

was not isolated until the early 1970s (Harris et al. 1972; Whiting et al. 1921). Since the late

2000s, several additional distinct taxa ('Brachyspira hampsonii' and 'Brachyspira suanatina') have

been found to causally associated with this syndrome (Burrough 2017). Swine dysentery, which

primarily affects grower-finisher pigs, is the most economically damaging disease associated

with Brachyspira attributable to both mortality and production limiting sub-optimal feed

conversion (Alvarez-Ordonez et al. 2013). Brachyspira pilosicoli has a broad host spectrum

including pigs, other domestic animals, wildlife and humans (Trivett-Moore et al. 1998). In pigs,

intestinal spirochetosis caused by Brachyspira pilosicoli typically affects pigs within 7 to 14

days post-weaning (Trott et al. 1996). Porcine intestinal spirochetosis is characterized by

diarrhea often described as having a "wet cement" consistency (Hampson and Duhamel 2006). In

chickens, Brachyspira intermedia and pilosicoli are associated with diarrhea, decreased

production and fecal staining of eggs (Mappley et al. 2014). Finally, human intestinal

spirochetosis caused by B. pilosicoli or B. aalborgi, is an infrequent cause of diarrhea most

commonly affecting children and immunocompromised individuals (Helbling et al. 2012;

Tateishi et al. 2015).

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Since the late 2000s the re-emergence of Brachyspira associated disease in pigs in North

America and the emergence of 'Brachyspira hampsonii' in Europe have been described (Mahu et

al. 2014; Martinez-Lobo et al. 2013; Rubin et al. 2013a). This re-emergence has included the

identification of novel taxa causing a dysentery like disease ('Brachyspira hampsonii' and

'Brachyspira suanatina') which have been effectively but not validly described (Chander et al.

2012; Mirajkar et al. 2016b; Mushtaq et al. 2015; Perez et al. 2016). The specific etiological

diagnosis of Brachyspira associated disease is challenging and relies heavily on PCR and culture

based assays; it was therefore not surprising that the emergence of 'B. hampsonii' has been

associated with substantial diagnostic challenges. The economic and animal welfare implications

of Brachyspira associated disease have led to renewed interest in improving the diagnostic

methods used for Brachyspira, including antimicrobial susceptibility test methods to facilitate

the evidence based use of antimicrobials. The purpose of this review is therefore to summarize

the current state of knowledge regarding the susceptibility of Brachyspira to antimicrobials, the

intrinsic methodological limitations associated with Brachyspira and to highlight future research

avenues to improve the reproducibility and reliability of Brachyspira antimicrobial susceptibility

testing.

2.0 General information about Brachyspira

2.1 Taxonomy

The genus Brachyspira is within the order Brachyspirales, and was recognized as a distinct

genus from Treponema in 1983 (Gupta et al. 2013; Hovind-Hougen et al. 1982; Oren and Garrity

2014). There are currently seven species with standing in the nomenclature: Brachyspira

hyodysenteriae, B. pilosicoli, B. murdochii, B. innocens, B. intermedia, B. aalborgi and B.

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alvinipulli (Euzeby 1997; Parte 2017). Brachyspira require specialized 'non-standard' culture

conditions, don't typically from colonies on agar, grow unpredictably in broth and are poorly

characterized phenotypically compared to other organisms such as E. coli; accurate etiological

identification is therefore challenging. In the 1970s pathogenic Brachyspira (then Treponema

hyodysenteriae) were identified as strongly hemolytic on blood containing media while weakly

hemolytic spirochetes were considered to be non-pathogens (Kinyon et al. 1977). The

identification of multiple, phylogenetically distinct weakly hemolytic Brachyspira highlights the

limitations of this approach. Molecular tools have proven to be extremely useful diagnostically,

although care must be taken to interpret test results considering the limitations of the test such as

primer sensitivity and specificity. Recently, MALDI-TOF has been explored for the

identification of Brachyspira species and shows promise (Calderaro et al. 2013; Prohaska et al.

2014; Warneke et al. 2014). Identification by phylogenetic analysis, particularly of the NADH

oxidase (nox) gene has proven useful and provides comparable, objective data which is portable

between labs. Sequence analysis at present is considered the gold standard for species level

identification (Perez et al. 2016; Rohde et al. 2014). DNA sequencing has revealed phylogenetic

diversity within the genus Brachyspira, including the novel strain 'B. hampsonii', and a large

number of other strains which have not yet been characterized but appear to be distinct from

recognized species (Patterson et al. 2013; Rubin et al. 2013b).

2.2 Treatment

In pigs Brachyspira associated disease has been effectively treated with the pleuromutilins,

macrolides/lincosamides and carbadox while metronidazole has been effectively used in people

with intestinal spirochetosis (Hampson 2012; Helbling et al. 2012). However, the ability to use

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some of these agents in food producing animals is restricted in many jurisdictions; metronidazole

is banned for use in food animals in North America and the European Union, and carbadox is

banned in Canada, the EU and the UK and recently had its approval rescinded in the United

States (FDA 2016). The pleuromutilins (tiamulin and valnemulin) and the

macrolide/lincosamides (tylosin, lincomycin and tylvalosin) are the mainstay of anti-Brachyspira

therapy in swine medicine, although a number of other antimicrobial including bacitracin,

virginiamycin and gentamicin are labeled for the treatment or prevention of Brachyspira

associated diseases in pigs (Table 1) (Hampson 2012). The heavy reliance on mechanistically

similar drugs, to which resistance in other organisms has been shown to develop by a common

mechanism highlights the potential impact of resistance emergence and the clinical value of

laboratory test guided therapeutic selection. Although there is no consensus method for

determining the antimicrobial susceptibility of Brachyspira, trends of decreasing susceptibility

among B. hyodysenteriae for the macrolides and pleuromutilins have been described in Europe

and the United States (Aarestrup et al. 2008; Lobova et al. 2004; Mirajkar et al. 2016a; Prasek et

al. 2014; Rugna et al. 2015).

3.0 Antimicrobial susceptibility testing

3.1 Importance of susceptibility testing to the prudent use of antimicrobials

3.1.1 Antimicrobial resistance

The term "antimicrobial resistance" is often used imprecisely. The categorization of an organism

as susceptible or resistant based on an in vitro test is designed to be clinically predictive, viewing

bacterial drug susceptibility from the patient's perspective. Susceptibility indicates a high

likelihood of clinical success, while resistance suggests a low likelihood of treatment success.

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Clinical breakpoints are therefore necessarily predicated on assumptions including the species

being treated, target pathogen, site of infection and treatment regimen (dose, dosing frequency

and route of administration) (Dalhoff et al. 2009; Mouton et al. 2012).

Bacterial drug resistance can be intrinsic or acquired, and mechanistically falls into one of four

broad categories: (i) enzymatic inactivation of the antibiotic (ii) decreased permeability of the

cell wall (iii) active efflux of the antimicrobial from within the cell and (iv) alteration or absence

of drug binding sites (Boerlin and White 2013). Intrinsic resistance is constitutive to the

physiology of a particular organism, examples include penicillin resistance among

Enterobacteriaceae and cephalosporin resistance in Enterococcus spp. Conversely, organisms

with a susceptible wild-type can become resistant as a result of chromosomal mutation or the

acquisition of resistance genes through transformation, transduction or conjugation (Boerlin and

White 2013).

3.1.2 Importance of susceptibility testing for resistance surveillance

Beyond the clinical applicability of antimicrobial susceptibility test results, there is value in

collecting this data for resistance surveillance. The purpose of resistance surveillance is to

identify temporal changes in antimicrobial susceptibility and to detect the emergence of novel

resistance phenotypes or genotypes. Globally, harmonization of test methodology, drug panels

tested and interpretive criteria, is recognized as a critical step to ensure that data generated in

different laboratories is comparable (OIE 2003). Substantial obstacles facing the detection of

emerging resistance in Brachyspira therefore include the lack of standardized test methods and

interpretive criteria, and consensus regarding which drugs should be included in test panels and

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what concentrations should be tested. The description of the emergence of resistance to anti-

Brachyspira therapies has at best relied upon small scale, geographically limited, single

laboratory studies, and at worse the spurious comparison of data generated using different

methods.

Epidemiological cut-offs based on distributions of MICs of bacterial populations, may prove to

be useful for resistance surveillance. Epidemiological cut-offs, based on MIC distributions of

bacterial populations, are useful for detecting organisms with acquired resistance that

differentiates them from the wild type population but does not address questions of clinical

efficacy because they do not consider host factors (Dalhoff et al. 2009).

3.2 General information on susceptibility testing methods

Early in the antibiotic era, the development of antimicrobial susceptibility test methods grew

from the need to select the most appropriate therapy in the face of emerging resistance and the

availability of new, mechanistically distinct compounds. The requirement for high quality

information led to the standardization of test methods which improved comparability of results

between labs, and allowed the development of uniform interpretive criteria (when to classify an

isolate as susceptible or resistant) (Ericsson and Sherris 1971). Regularly updated internationally

recognized standardized methods are now published by the Clinical and Laboratory Standards

Institute (CLSI) in the United States, and the European Committee on Antimicrobial

Susceptibility Testing (EUCAST) in Europe. Because test results vary with divergences from

standard methodologies, test guidelines are necessarily prescriptive in terms of the density and

growth phase of the test culture, test media composition including pH and cation concentration,

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incubation temperature, time and atmosphere and endpoint definition (CLSI 2012). The effects

of test chemistry on MIC are well recognized but vary by drug and organism. For example test

media with too low a pH may yield decreased penicillin MICs or increased quinolone

MICs(CLSI 2017). Other test factors are intuitively more universal; for instance low MICs may

be an artifact of too low a test inoculum, while high MICs result from testing too many

organisms (CLSI 2017). Although test methods specific to a wide variety of anaerobic and

fastidious organisms (ex. HACEK group organisms, Helicobacter, Listeria, Moraxella,

Campylobacter, Actinobacillus pleuropneumoniae) are available, no standardized protocols

(human or veterinary) addressing the unique requirements of Brachyspira have been published

(CLSI 2015, 2016, 2017). The methods described by the CLSI and EUCAST all rely on

phenotypic indicators of susceptibility, either growth or inhibition of growth in the presence of

antimicrobial. Unambiguous and standardized endpoint definition is therefore essential (Jenkins

and Schuetz 2012).

Molecular techniques (PCR) are increasingly used to identify the presence of resistance genes

and therefore infer resistance. PCR has the advantage of detecting resistance without requiring

classical phenotypic susceptibility test methods, a potential benefit when characterizing

Brachyspira. However, the use of molecular methods requires knowledge of resistance genes or

resistance conferring mutations. It must also be recognized that while these methods can identify

resistance, they are insufficient to confirm susceptibility due to the potential presence of novel

resistance genes or resistance conferring mutations not included in the assay.

4.0 Specific challenges to susceptibility testing of Brachyspira

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The ability to assess the antimicrobial susceptibility of Brachyspira is hindered by two proximate

causes: 1. the growth conditions required for Brachyspira are different than those standardized

for susceptibility testing and 2. the inability to use typical susceptibility test endpoints. Although

Brachyspira are reported to grow within the range of conditions prescribed by the CLSI for

antimicrobial susceptibility testing, no single set of conditions have been standardized for testing

these organisms. The unusual growth characteristics of Brachyspira sp. are at the root of many of

these challenges. On solid media Brachyspira do not form distinct colonies, growth is typically

inferred by the presence of hemolytic zones on blood containing agar (Figure 1). This property

complicates/precludes the application of the concept of the colony forming unit, a foundational

bacteriological principle which allows viable cells to be enumerated, and genetic clones to be

isolated. The inability to differentiate whether apparently distinct hemolytic zones represent

unique founding organisms or multiple individuals is a critical limitation. Pure cultures are a pre-

requisite for antimicrobial susceptibility testing, tests of mixed cultures yield cumulative results

which may not reflect the susceptibility of either organism individually. Furthermore, this

characteristic potentially precludes reliable test endpoint definition; whereas an antimicrobial

inhibiting the formation of a colony is taken to indicate growth inhibition, failure to observe

haemolysis in the presence of an antimicrobial may simply reflect inhibition of haemolysin

production with or without affects on microbial growth. This is particularly germane for

Brachyspira where protein synthesis inhibiting drugs are the primary agents of interest. Creative

approaches to address endpoint validity including microscopic evaluation or sub-culture of

media may prove useful in identifying viable organisms in media containing supra-MIC drug

concentrations.

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Broth culture of Brachyspira spp. also presents some unique challenges. Anecdotal observations

suggest that broth cultures require a high starting inoculum and so failure to grow an organism in

broth may simply reflect an insufficient initial inoculum density. The complicates two steps of

the susceptibility testing process: 1. growing an initial broth culture used to inoculate test media,

and 2. interpreting broth based susceptibility tests - did the culture not grow because it was

inhibited by the drug, or because the culture was insufficiently dense?

5.0 Summary of susceptibility test methods which have been used for Brachyspira

The English language literature through 2017 was searched and studies describing antimicrobial

susceptibility testing of Brachyspira were reviewed. Both agar and broth dilution techniques

were used although there was substantial variability in the methods reported (Table 2).

Incubation temperatures ranged from 37 – 42 °C with incubation times ranging from 2 to 5 days

for agar dilution, and 37 – 38 °C and 3-5 days for broth dilution respectively. Where reported,

highly variable inoculum densities were described:1 X 105

- 5 X 106 CFU/ml for broth dilution

and 104

- 106 CFU/spot for agar dilution tests. Finally, where reported atmospheres with varying

composition were used. According to the manufacturers all anaerobic systems yield a final O2

concentration of ≤1%, but yield highly variable CO2 concentrations (7- >15%). A 2016 study has

also described the use of doxycycline gradient strips although this method is not widely used

(Mirajkar and Gebhart 2016). Further complicating the interpretation of susceptibility test

results, is the lack of agreed upon interpretive criteria. With the lack of CLSI and EUCAST

resistance breakpoints, investigators have relied upon a number of divergent sets of researcher

proposed breakpoints (Burch 2005; Duhamel et al. 1998; Pringle et al. 2012; Ronne and Szancer

1990; SVARM 2014).

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The introduction of the VetMIC Brachyspira broth micro-dilution system, first described in

2001, has led to substantial progress towards test standardization (Karlsson et al. 2004a;

Karlsson et al. 2001). This product includes serial two-fold dilutions of dried antimicrobials in

tissue culture trays. Testing is done by inoculating wells with 500µl of broth culture and

incubating anaerobically at 37⁰C for 4 days with agitation (SVA 2011). Antimicrobial MICs as

determined using this method have been shown to be reproducible, although interestingly

typically yield a doubling dilution lower MIC than agar dilution tests (Rohde et al. 2004).

6.0 Challenges in comparing susceptibility data generated using non-standard tests

Because, there are no established standard test methods, interpretive criteria comparison of

results generated by different labs must only be done with extreme caution. This inability to

compare data was exemplified by a 2005 study where eight European laboratories participated in

a ring test of Brachyspira diagnostics (Rasback et al. 2005). Each laboratory received samples of

pig feces seeded with a previously identified Brachyspira isolate at specific concentrations and

two pure cultures of known Brachyspira for susceptibility testing. Antibiotic susceptibility test

methods differed by media composition and type, incubation temperature and time. Only a very

limited description of the methods used by each laboratory for susceptibility testing and

interpretation was presented. Not surprisingly results were highly variable; errors including

failure to detect or identify isolates or discordant susceptibility test results occurred 32% of the

time (Rasback et al. 2005). The varying concentration of antimicrobials tested made comparison

of MICs between labs impossible even when the results were not inconsistent (ex. different labs

reported lincomycin MIC of ≤4, ≤2 and ≤0.5 for the same isolate). The lack of validated

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Brachyspira specific susceptibility test methods has resulted in the development of a variety of

"in-house" techniques, consequently susceptibility data are not reliably comparable between labs.

Further confounding susceptibility data portability is the lack of a consensus scheme for

determining species level identification (Rasback et al. 2005). Researchers have used

combinations of conventional methods (ex. culture and biochemical tests) and a variety of

molecular methods (ex. nox and 16S-rDNA sequencing and nox-RFLP) which are recognized to

have varying levels of sensitivity and specificity.

7.0 Objective measures of resistance

Genetic associations with decreased susceptibility to a number of drugs have been elucidated in

several Brachyspira spp. (Table 3). Most published studies have focused on the association

between single nucleotidepolymorphisms (SNPs) in genes encoding ribosomal RNA and

ribosomal proteins and increased MIC to drugs which act by inhibiting protein synthesis

inhibitors including macrolides, lincosamides, pleuromutilins, doxycycline, chloramphenicol and

florfenicol (Hidalgo et al. 2011; Hillen et al. 2014; Pringle et al. 2007; Pringle et al. 2004;

Verlinden et al. 2011). Narrow spectrum β-lactamases including 13 enzymes related to OXA-63

have also been identified in B. pilosicoli (La et al. 2015; Meziane-Cherif et al. 2008; Mortimer-

Jones et al. 2008). Differences in intrinsic resistance, even between closely related bacteria can

inform treatment decisions based on accurate organism identification. For example, differences

in the intrinsic resistance to vancomycin of Enterococcus gallinarum but not Enterococcus

faecalis or faecium (CLSI 2017). Among Brachyspira species differences in susceptibility have

also been observed, one study reported significant differences between species (Clothier et al.

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2011). Although this study was small, it highlights the possibility of identifying taxa-specific

differences in intrinsic resistance which may prove useful to clinicians for therapeutic selection.

8.0 Summary and future directions

The lack of standardized antimicrobial susceptibility test methods and interpretive criteria for

Brachyspira is an important limitation to the evidence based use of antimicrobials for treating

clinical disease. These diagnostic limitations have been made acutely problematic by the re-

emergence of Brachyspira associated disease in North America, and the emergence of novel taxa

in Europe. Furthermore, the increasing public scrutiny of antimicrobial usage in agriculture

provides constant pressure to ensure that prescribing practices are evidence based and prudent.

Unfortunately, for the reasons described here, we do not recommend that diagnostic laboratories

routinely report categorical susceptibility test results for Brachyspira spp. Instead, we encourage

our colleagues to continue their research describing temporal changes in MIC and archiving

Brachyspira culture collections to facilitate the eventual development of agreed upon test

methodology and interpretive criteria.

The lack of consensus on how to identify an isolate to the species level is the most immediate

obstacle to improving laboratory diagnostics for Brachyspira. A common ontology is critical for

researchers and diagnosticians to make simple comparisons of laboratory prevalence, describe

the distribution of particular taxa or to compare the drug susceptibility of two organisms. The

next most urgent need is an enriched understanding the genetic determinants of resistance. These

genes or mutations have the potential to serve as an objective data point against which

phenotypic susceptibility can be benchmarked, potentially serving as a pillar of quality control.

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Finally, we call for additional collaboration in the global Brachyspira diagnostic/research

communities. Collaboration and consensus building is going to be a critical step in identifying

and recommending best practices for Brachyspira. This leadership was exemplified by Rasback

et al., 2005 where the results of a multi-centre ring test were described; such investigations will

be essential not only for ensuring method portability and objectivity, but also as a feedback

mechanism to identify areas for improvement in individual laboratories.

Acknowledgments

The authors would like to thank Elanco (formerly Novartis Animal Health) and Agriculture and

Agri-Food Canada through Swine Innovation Porc for funding this investigation and providing

stipend support for DGRSK. Stipend support from the Department of Veterinary Microbiology

devolved fund is also appreciated. We would like to acknowledge Mo Patterson for the

photograph depicted in Figure 1. Finally the authors would like to thank the veterinarians and

producers of the Canadian swine industry for their continued collaboration and support of our

research.

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Figure 1.

Hemolytic zones associated with the growth of Brachyspira hyodysenteriae ATCC 27164 on CVS agar.

Note the lack of colony formation on the surface of the media.

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Table 1. Antimicrobials with label claims for the treatment/prevention/control of Brachyspira associated disease

Drug Name United States Dosage* Canada Dosage‡ European Union Dosage‡

Tylosin YES Water - 250mg/gallon

Feed - 40-100g/kg feed YES Feed - 44-110 mg/kg NO

Lincomycin YES Feed - 40-100g/kg YES

Feed - 44-110 mg/kg NO

Tylvalosin NO NO YES Water - 5mg/kg

Feed - 4.25mg/kg

Tiamulin YES Feed - 200g/ton YES Feed - 31.2-220.1 mg/kg NO

Valnemulin NO NO YES Feed - 1-4mg/kg

Viriginiamycin YES Feed - 25-100g/ton YES Feed - 55-110 mg/kg NO

Carbadox NO NO NO

Bacitracin YES Feed - 250g/ton NO NO

Gentamicin YES Water - 50mg/gallon NO NO

*administered dosage is per mass or volume of feed or warer, ‡ administered dosage is per mass of pig receiving medication

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Table 2. Chronologicakl summary of antimicrobial susceptibility test methodologies utilized in published investigations

Method Country Year Organism Incubation Temp Incubation Time Final Inoculum Atmosphere Media References

Ag

ar

Dil

uti

on

Sweden 2003 Various 37⁰C 96 hours 1 -5 x 105 / spot GENbox TSA5OX

WC5SB

(Karlsson et al. 2003)

Japan 2004 BH Unclear, 37-38⁰C 72 hours 104 - 10

5 / spot BBD GasPak TSA5SB (Uezato et al. 2004)

Czech

Republic

2004 BH 37⁰C 72 - 96 hours 104 / spot Anaerobic jar using gas

generating kit (BR38, Oxoid)

WC5SB (Lobova et al. 2004)

Germany 2004 BH 42⁰C 48 hours 3X 105 / spot No details reported TSA10BB (Rohde et al. 2004)

Japan 2010 BH 37⁰C 72 hours 105 / spot No details reported TSA5SB (Ohya and Sueyoshi 2010)

USA 2011 Various 42⁰C 48-96 hours 1.6 - 4 x 104 / spot GasPak TSA5SB (Clothier et al. 2011)

Korea 2012 Various 37⁰C 72 - 120 hours 1.5 x 106 / spot No details reported MHA5SB (Lim et al. 2012)

Czech

Republic

2011 BH 37⁰C 72-96 hours Undefined AnaeroGen WC5SB (Sperling et al. 2011)

Czech

Republic

2014 BH 37⁰C 72-96 hours 105 / spot AnaeroGen WC5SB (Prasek et al. 2014)

Japan 2015 BH 37⁰C 72 hours 104 - 10

5 / spot No details reported TSA5SB (Kajiwara et al. 2015)

USA 2016 Various 37⁰C 96 hours 1 x 105 / spot No details reported TSA5SB0 (Mirajkar et al. 2016a)

Bro

th D

ilu

tio

n

Sweden 1999 BH 37⁰C up to 96 hours 5 x 105 - 10

6 CFU/ml No details reported BHIS10 (Karlsson et al. 1999)

Sweden §2001 BH 37⁰C 96 hours 1 - 5 x 106

CFU/ml No details reported BHIS10 (Karlsson et al. 2001)

Australia 2002 BH 37⁰C 96 hours 1 - 5 x 106 CFU/ml GENbox BHIS10 (Karlsson et al. 2002)

Sweden §2003 Various 37⁰C 96 hours 1 - 5 x 106 CFU/ml GENbox BHIS10 (Karlsson et al. 2003)

Sweden §2004 BP 37⁰C 96 hours 1 - 5 x 106 CFU/ml GENbox BHIS10 (Karlsson et al. 2004b)

Germany 2004 BH 37⁰C 96 hours 3 × 106

CFU/ml GENbox BHIS10 (Rohde et al. 2004)

European §2004 BH 37⁰C 96 hours 1 - 5 x 106 CFU/ml GENbox BHIS10 (Karlsson et al. 2004a)

Sweden §2006 BP 37⁰C 96 hours 1 - 5 x 106 CFU/ml BBD GasPak BHIS10 (Pringle et al. 2006)

Spain §2009 BH 38⁰C 72-120 hours 1 - 5 x 106 CFU/ml GENbox BHIS10 (Hidalgo et al. 2009)

Spain §2011 BH 37⁰C 96 hours 1 - 5 x 106 CFU/ml No details reported BHIS10 (Hidalgo et al. 2011)

Sweden §2012 BH, BP 37⁰C 96 hours 1 - 5 × 106 CFU/ml No details reported BHIS10 (Pringle et al. 2012)

Poland §2012 BH 37⁰C 96 hours 106

CFU/ml GENbox BHIS10 (Zmudzki et al. 2012)

Germany 2014 BH 37⁰C 120 hours 105

GFU50/ml*

GENbox BHIS20 (Herbst et al. 2014)

Italy §2015 BH No details reported (Rugna et al. 2015)

USA §2016 Various 37⁰C 96 hours 106

CFU/ml No details reported BHIS10 (Mirajkar et al. 2016a)

BH - B. hyodysenteriae; BP - B. pilosicoli

Media: Tryptic soy agar + 5% sheep blood: TSA 5 SB; Tryptic soy agar + 10% sheep blood: TSA 10 SB; Tryptic soy agar + 5% ox blood: TSA 5 OX; Tryptic soy agar + 10% bovine blood: TSA10BB; Wilkins-

Charlgren + 5% sheep blood: WC 5SB; Mueller-Hinton agar + 5% sheep blood: MHA 5 SB; Brain heart infusion + 10% fetal calf serum: BHIS 10; Brain heart infusion + 20% fetal calf serum: BHIS 20;

Atmosphere: AnaeroGen Sachets Oxoid: AnaeroGen; Anaerobic gas pack GasPak BD: GasPak; anaerobic atmosphere generator biomerieux GENbox: GENbox; Anaerobic gas generator envelopes BBL

gas pak: BBD GasPak

*GFU - authors report "growth forming units" as opposed to CFU§Studies which reported the use of the VetMIC Brachy panel

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Table 3. Genetic associations with decreased antimicrobial susceptibility in Brachyspira

Organisms

Investigated

Phenotypic

Resistance

Genetic Mechanism References

B. hyodysenteriae

B. pilosicoli

macrolides,

lincosamides,

streptogramins,

tiamulin

Single nucleotide

polymorphisms at positions

2032 and 2058 of the 23S

ribosomal subunit

(Hidalgo et al. 2011;

Karlsson et al. 1999;

Karlsson et al.

2004b)

B. hyodysenteriae tiamulin Mutation in ribosomal

protein L2, L3, L4, L22 and

23S rRNA in the peptidyl

transferase region

(Hillen et al. 2014;

Pringle et al. 2004)

B. hyodysenteriae

B. intermedia

doxycycline Single nucleotide

polymorphism at position

1058 of the 16S ribosomal

subunit

(Pringle et al. 2007;

Verlinden et al.

2011)

B. pilosicoli penicillin,

ampicillin and

oxacillin

β-lacatamases (OXA-63,

OXA-136, OXA-137 and OXA-

470 - OXA-479)

(La et al. 2015;

Meziane-Cherif et

al. 2008)

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Hemolytic zones associated with the growth of Brachyspira hyodysenteriae ATCC 27164 on CVS agar. Note the lack of colony formation on the surface of the media.

180x179mm (300 x 300 DPI)

Page 31 of 31



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